ABSTRACT Although no animal model exactly duplicates clinically defined alcoholism, models for specific factors, such as the withdrawal syndrome, are useful for identifying potential neural determinants of liability in humans. The well-documented difference in withdrawal severity following chronic ethanol exposure, between the DBA/2J and C57BL/6J mouse strains, provides an excellent starting point for dissecting the neural circuitry affecting predisposition to physical dependence on ethanol. To induce physical dependence, we used a paradigm in which mice were continuously exposed to ethanol vapor for 72h. Ethanol-exposed and air-exposed (control) mice received daily injections of pyrazole hydrochloride, an alcohol dehydrogenase inhibitor, to stabilize blood ethanol levels. Ethanol-dependent and air-exposed mice were killed 7h after removal from the inhalation chambers. This time point corresponds to the time of peak ethanol withdrawal severity. The brains were processed to assess neural activation associated with ethanol withdrawal indexed by c-Fos immunostaining. Ethanol-withdrawn DBA/2J mice showed significantly (P<.05) greater neural activation than ethanol-withdrawn C57BL/6J mice in the dentate gyrus, hippocampus CA3, lateral septum, basolateral and central nuclei of the amygdala, and prelimbic cortex. Taken together with results using an acute model, our data suggest that progression from acute ethanol withdrawal to the more severe withdrawal associated with physical dependence following chronic ethanol exposure involves recruitment of neurons in the hippocampal formation, amygdala, and prelimbic cortex. To our knowledge, these are the first studies to use c-Fos to identify the brain regions and neurocircuitry that distinguish between chronic and acute ethanol withdrawal severity using informative animal models.

[Show abstract][Hide abstract]ABSTRACT: Background
The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP; allopregnanolone) has been studied during withdrawal from ethanol (EtOH) in humans, rats, and mice. Serum 3α,5α-THP levels decreased, and brain levels were not altered following acute EtOH administration (2 g/kg) in male C57BL/6J mice; however, the effects of chronic intermittent ethanol (CIE) exposure on 3α,5α-THP levels have not been examined. Given that CIE exposure changes subsequent voluntary EtOH drinking in a time-dependent fashion following repeated cycles of EtOH exposure, we conducted a time-course analysis of CIE effects on 3α,5α-THP levels in specific brain regions known to influence drinking behavior.Methods
Adult male C57BL/6J mice were exposed to 4 cycles of CIE to induce EtOH dependence. All mice were sacrificed and perfused at 1 of 2 time points, 8 or 72 hours following the final exposure cycle. Free-floating brain sections (40 μm; 3 to 5 sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3α,5α-THP.ResultsWithdrawal from CIE exposure produced time-dependent and region-specific effects on immunohistochemical detection of 3α,5α-THP levels across cortical and limbic brain regions. A transient reduction in 3α,5α-THP immunoreactivity was observed in the central nucleus of the amygdala 8 hours after withdrawal from CIE (−31.4 ± 9.3%). Decreases in 3α,5α-THP immunoreactivity were observed 72 hours following withdrawal in the medial prefrontal cortex (−25.0 ± 9.3%), nucleus accumbens core (−29.9 ± 6.6%), and dorsolateral striatum (−18.5 ± 6.0%), while an increase was observed in the CA3 pyramidal cell layer of the hippocampus (+42.8 ± 19.5%). Sustained reductions in 3α,5α-THP immunoreactivity were observed at both time points in the lateral amygdala (8 hours −28.3 ± 12.8%; 72 hours −27.5 ± 12.4%) and in the ventral tegmental area (8 hours −26.5 ± 9.9%; 72 hours −31.6 ± 13.8%).Conclusions
These data suggest that specific neuroadaptations in 3α,5α-THP levels may be present in regions of brain that mediate anxiety, stress, and reinforcement relevant to EtOH dependence. The changes that occur at different time points likely modulate neurocircuitry involved in EtOH withdrawal as well as the elevated drinking observed after CIE exposure.

[Show abstract][Hide abstract]ABSTRACT: Ethanol abuse can lead to addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. There is evidence for contributions of both genotype and sex to alcoholism, but an understanding of the biological underpinnings is limited. Utilizing both sexes of genetic animal models with highly divergent alcohol withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) mice, the distinct contributions of genotype/phenotype and of sex during addiction stages on neuroadaptation were characterized. Transcriptional profiling was performed to identify expression changes as a consequence of chronic intoxication in the medial prefrontal cortex. Significant expression differences were identified on a single platform and tracked over a behaviorally-relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more sensitive to ethanol with higher fold expression differences. Bioinformatics showed a strong effect of sex on the data structure of expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, differences were observed instead between the lines/phenotypes irrespective of sex. Confirmation of identified pathways showed distinct inflammatory signaling following intoxication at peak withdrawal, with a pro-inflammatory phenotype in females but overall suppression of immune signaling in males. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of stage- and sex-specific therapies for alcohol withdrawal and the maintenance of sobriety.

[Show abstract][Hide abstract]ABSTRACT: Chronic alcohol abuse depresses the nervous system and, upon cessation, rebound hyperexcitability can result in withdrawal seizure. Withdrawal symptoms, including seizures, may drive individuals to relapse, thus representing a significant barrier to recovery. Our lab previously identified an upregulation of the thalamic T-type calcium (T channel) isoform CaV3.2 as a potential contributor to the generation and propagation of seizures in a model of withdrawal. In the present study, we examined whether ethosuximide (ETX), a T-channel antagonist, could decrease the severity of ethanol withdrawal seizures by evaluating electrographical and behavioral correlates of seizure activity. DBA/2J mice were exposed to an intermittent ethanol exposure paradigm. Mice were treated with saline or ETX in each withdrawal period, and cortical EEG activity was recorded to determine seizure severity. We observed a progression in seizure activity with each successive withdrawal period. Treatment with ETX reduced ethanol withdrawal-induced spike and wave discharges (SWDs), in terms of absolute number, duration of events, and contribution to EEG power reduction in the 6–10 Hz frequency range. We also evaluated the effects of ETX on handling-induced convulsions. Overall, we observed a decrease in handling-induced convulsion severity in mice treated with ETX. Our findings suggest that ETX may be a useful pharmacological agent for studies of alcohol withdrawal and treatment of resulting seizures.

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Differential activation of limbic circuitry associated with chronicethanol withdrawal in DBA/2J and C57BL/6J miceGang Chen1,2,*, Matthew T. Reilly1,2,*,#, Laura B. Kozell1,2, Robert Hitzemann1,2,3, and KariJ. Buck1,2,31 Department of Behavioral Neuroscience, Veterans Affairs Medical Center, Portland, OR 972392 Portland Alcohol Research Center, Veterans Affairs Medical Center, Portland, OR 972393 Oregon Health & Science University, Portland, OR 97239AbstractAlthough no animal model exactly duplicates clinically defined alcoholism, models for specificfactors, such as the withdrawal syndrome, are useful for identifying potential neural determinants ofliability in humans. The well-documented difference in withdrawal severity following chronicethanol exposure, between the DBA/2J and C57BL/6J mouse strains, provides an excellent startingpoint for dissecting the neural circuitry affecting predisposition to physical dependence on ethanol.To induce physical dependence, we used a paradigm in which mice were continuously exposed toethanol vapor for 72 hr. Ethanol-exposed and air-exposed (control) mice received daily injections ofpyrazole hydrochloride, an alcohol dehydrogenase inhibitor, to stabilize blood ethanol levels.Ethanol-dependent and air-exposed mice were killed seven hours after removal from the inhalationchambers. This time point corresponds to the time of peak ethanol withdrawal severity. The brainswere processed to assess neural activation associated with ethanol withdrawal indexed by c-Fosimmunostaining. Ethanol-withdrawn DBA/2J mice showed significantly (p<0.05) greater neuralactivation than ethanol-withdrawn C57BL/6J mice in the dentate gyrus, hippocampus CA3, lateralseptum, basolateral and central nuclei of the amygdala, and prelimbic cortex. Taken together withresults using an acute model, our data suggest that progression from acute ethanol withdrawal to themore severe withdrawal associated with physical dependence following chronic ethanol exposureinvolves recruitment of neurons in the hippocampal formation, amygdala and prelimbic cortex. Toour knowledge, these are the first studies to use c-Fos to identify the brain regions and neurocircuitrythat distinguish between chronic and acute ethanol withdrawal severity using informative animalmodels.KeywordsEthanol; c-Fos; Hippocampus; Amygdala; Limbic; WithdrawalCorresponding Author: Gang Chen, Ph.D., 3710 US Veterans Hospital Road, Research Services (mailcode R&D40), Portland, OR97239-3098, chenga@ohsu.edu, Phone: 503-220-8262, ext. 54145, Fax: 503-220-3411.*Co-first authors#Current address: Division of Neuroscience & Behavior, National Institute on Alcohol Abuse & Alcoholism, Bethesda, MD 20892-1705Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptAlcohol. Author manuscript; available in PMC 2010 September 1.Published in final edited form as:Alcohol. 2009 September ; 43(6): 411–420. doi:10.1016/j.alcohol.2009.05.003.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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IntroductionWithdrawal hyperexcitability of the central nervous system (CNS) is a well-knownconsequence of physical dependence on ethanol, and is one of the most alarming manifestationsof the withdrawal syndrome in alcoholics. During extended ethanol exposure, compensatory(homeostatic) changes occur in the CNS leading to the development of functional tolerance inthe presence of ethanol, as well as to cross-tolerance to other depressant drugs. When theethanol is withdrawn (i.e., its use is discontinued), these previously compensatoryneuroadaptive processes exhibit overcompensation (or rebound), leading to the CNShyperexcitability signs that are characteristic of the withdrawal syndrome (Littleton, 1998).Symptoms of ethanol withdrawal in animals are similar to those seen in humans, and includetremor, other motoric dysfunctions, autonomic overactivity and, in the most severe cases,seizures (De Witte et al., 2003).The brain region(s) and circuit(s) involved in naturally-occurring, genetically-determineddifferences in neural activation associated with ethanol withdrawal are not known. Immediateearly gene product expression has proven useful to assess the pattern of neural activation inethanol withdrawn rodents following chronic ethanol exposure (Le et al., 1992; Morgan et al.,1992; Moy et al., 2000; Olive et al., 2001; Wilce et al., 1994). However, only one of thesestudies assessed genetically determined differences in neural activation and compared proteinkinase C-ε (PKCε) knockout and wild-type PKCε+/+ mice (Olive et al., 2001). This comparisonidentified seven brain regions potentially involved in genetically determined differences inchronic ethanol withdrawal severity (i.e., the lateral septum, substantia nigra, dentate gyrus,paraventricular nuclei of the hypothalamus and thalamus, and mediodorsal nucleus of thethalamus), but it is not known to what degree these results are specific to the comparison ofPKCε−/− and wild-type mice and to what extent the results may generalize to more widely usedanimal models. The DBA/2J (D2) and C57BL/6J (B6) mouse strains are the most widelystudied animal models of severe and mild ethanol withdrawal, respectively, but have notpreviously been compared for neural activation following withdrawal from chronic ethanol.Importantly, mapping populations derived from the D2 and B6 progenitor strains have beenused successfully to begin to dissect the genetic determinants of physical dependence andassociated withdrawal episodes following chronic ethanol exposure (Buck et al., 2002; Crabbe,1998). Identification of the neural determinants that differentiate withdrawal between thesetwo informative strains therefore offers the distinct advantage of identifying specific brainregion(s) and circuitry by which these genes exert their effects on ethanol dependence andassociated withdrawal.Another important goal of our research is to identify the brain region(s) and circuitry involvedin the progression from mild withdrawal associated with acute ethanol exposure to more severewithdrawal exhibited by physically dependent animals following chronic ethanol exposure.The most commonly used animal models of ethanol withdrawal include chronic models wherewithdrawal is apparent following exposure to ethanol vapor for 72 hr (Buck et al., 2002; Terdaland Crabbe, 1994) or a liquid diet containing ethanol for several days (Levental and Tabakoff,1980); and an acute model where withdrawal can be demonstrated after a single hypnotic doseof ethanol (Buck et al., 1997). The studies reported here use the chronic ethanol vapor modeland are compared to our results using an acute model (Kozell et al., 2005). We hypothesizedthat distinct brain regions within the basal ganglia and/or associated cortical and limbiccircuitry would exhibit genotype-dependent neuronal activation associated with chronicethanol withdrawal severity between the D2 and B6 mouse strains.Chen et al.Page 2Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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METHODSAnimalsMale B6 and D2 mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Uponarrival at the AALAC accredited Portland VA Medical Center, mice were housed in groups offour per cage with corn cob bedding and allowed to acclimate for at least one week beforebeing transferred to ethanol vapor inhalation chambers or identical control (air) chambers. Themice were 70–100 days old at the time of testing. Food (Purina LabDiet, Purina MillsInternational, St. Louis MO) and water were freely available at all times. Colony and procedurerooms were maintained at an ambient temperature of 21±1°C. All procedures were approvedby the institutional IACUC committee and followed guidelines for the care and use oflaboratory animals from the National Institutes of Health and the Department of VeteransAffairs.Induction of Ethanol Physical DependenceAll groups were treated in parallel and sacrificed on the same day. To induce physicaldependence on ethanol, mice were continuously exposed to ethanol vapor for 72 hr. Ethanol-exposed mice received a loading dose of 1.5 g/kg ethanol at the beginning of the ethanol vaporexposure to raise blood ethanol concentrations (BECs) to the level to be maintained duringexposure. Ethanol-exposed and air-exposed (control) mice received daily injections of pyrazolehydrochloride (68 mg/kg, ip), an alcohol dehydrogenase inhibitor, to stabilize blood ethanollevels and reduce variability among individuals and between strains, in BEC values. Bloodsamples were also taken at 24 and 48 hr to monitor BECs for adjusting vapor concentration, ifnecessary. After 72 hr, all the mice were removed from the vapor chambers (between 8:00–9:00 AM) and a 20 μl tail blood sample was collected from the ethanol dependent mice fordetermination of individual BEC values using gas chromatography (Terdal and Crabbe,1994). Control mice also had their tails nicked, but no blood sample was collected. The miceremained undisturbed in their home cages for 7 hr, when they were killed by cervicaldislocation. The brains were removed and placed in ice-cold 4% paraformaldehyde in 0.1Mphosphate buffer (PB) overnight. The following day this was replaced with ice-cold 30%sucrose-PB. When the brains no longer floated, they were processed for immunohistochemicalanalysis. The 7 hr time point was used to assess immediate early gene expression during chronicethanol withdrawal because withdrawal associated handling-induced convulsions (HICs) beginabout 4–5 hr post-ethanol exposure and peak in severity approximately 6–7 hr after ethanolexposure is terminated in the animal models used in the present study (Buck et al., 2002), andbecause c-Fos protein induction typically occurs within 1 hr of such stimuli (Chang et al.,1995; Morgan et al., 1987; Young et al., 1991).Immunohistochemical analysis of neural activationThe immunohistochemical procedure has been previously described (Kozell et al., 2005; Chenet al., 2008). Notably, the mice were not tested for ethanol withdrawal convulsions in order toavoid potential confounds of evoked convulsions on c-Fos immunoreactivity. The mice werekilled by cervical dislocation and the brain was removed and placed in ice-cold 4%paraformaldehyde in 0.1 M phosphate buffer (PB) overnight. The following day the 4%paraformaldehyde solution was replaced with 0.1 M PB containing 30% sucrose until the brainno longer floated and was processed for immunohistochemical analysis. Brains were coronallysectioned (30 μm) on a freezing microtome and the tissue was stored in 10 mM PB containing0.02% sodium azide until processing. All of the experimental groups were processed at thesame time. The sections were first rinsed three times in 10 mM PB before being incubated in1.5% hydrogen peroxide in 10 mM PB in 0.9% saline solution (PBS) for 15 min to blockendogenous peroxidase activity, and then washed six times in 10 mM PBS. Sections wereblocked for 2 hr in immunoreaction buffer (10 mM PBS containing 0.25% Triton-X 100 andChen et al. Page 3Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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5% dry milk) without antibody; and rabbit anti-c-Fos antibody (1:10,000; Oncogene ScienceInc., Cambridge, MA) was then added and the incubation was continued for 72 hr at 4°C. Thespecificity of this antibody for c-Fos has been confirmed (Chan and Sawchenko, 1995). Thesections were rinsed three times in 10 mM PBS and incubated with biotinylated goat anti-rabbitIgG (1:200; Vector Laboratories, Burlingame, CA) in 10 mM PBS. The sections weresubsequently incubated with horseradish peroxidase avidin-biotin complex in 10 mM PBS for1.5 hr at room temperature (ABC Elite peroxidase kit, Vector Laboratories, Burlingame, CA).The sections were rinsed three times in 10 mM PBS and placed in 0.05 M Tris (pH 7.4) for 5min. The chromatic reaction was completed with diaminobenzidine (50 mg/100 ml of 0.05 MTris, Sigma, St. Louis, MO) in the presence of 0.01% nickel ammonium sulfate solution and0.035% hydrogen peroxide. Omission of the primary antibody to the sections was used as astaining control. The sections were mounted onto slides, dehydrated, and cover-slipped inPermount (Fisher Scientific, Pittsburgh, PA).Methods for analysis of quantitative morphometric analysis of c-Fos positive cells in thedistinct brain regions have previously been described (Chen et al., 2008; Demarest et al.,1998; Hitzemann and Hitzemann, 1997). Briefly, an Olympus BX60 light microscope andLEICA DFC 480 imaging system were used to obtain a permanent record of cell distributionfor c-Fos quantification. In our experience, results using mean densities across a brain regionand representative sections are comparable (Chen et al., 2008), so representative sections wereanalyzed for each brain region as follows (from Paxinos and Franklin, 2001): the central,basolateral and medial nuclei of the amygdala (plate 43), substantia nigra (plate 57), entorhinalcortex and hippocampal regions (plate 49), cingulate and prelimbic cortices (plate 18), nucleusaccumbens core, shell and the dorsal lateral caudate putamen (plate 21), lateral and medialseptum (plate 26), bed nucleus of the stria terminalis (plate 30), primary auditory and sensorycortices (plate 52), granular and dysgranular insular cortex (plate 22), motor and piriformcortices (plate 21), lateral and medial parietal association (posterior parietal) cortex (plate 45),paraventricular nucleus of the hypothalamus (PVN; plate 42), anterior paraventricular thalamus(PVA, plate 35), and mediodorsal thalamus (plate 37). All images were taken at 10X and signalswere quantified using Image Pro Plus (Media Cybernetics), except for the caudate nucleus andnucleus accumbens, which were quantified at 4X. Brain sections at 4X are used to illustratedifferences between groups in hippocampal formation in Figure 2. Standardized brain regiontemplates based on established anatomical markers were employed. Standardized thresholdparameters (160, light intensity range from 0 to 255) were employed to identify and quantifyindividual c-Fos positive neurons. Sample size estimates were based on previous studies (Chenet al., 2008; Demarest et al., 1998; Hitzemann and Hitzemann, 1997; Kozell et al., 2005) andare sufficient to overcome potential artifacts associated with assessing representative sections(Hitzemann and Hitzemann, 1997). The experimenter was blind to the experimental conditionfor each subject.Data analysisFor comparisons of ethanol withdrawal associated c-Fos induction, the data were not normallydistributed based upon a significant Kolmogorov-Smirnov test. Therefore, analysis of the datawith a standard analysis of variance (ANOVA) is not appropriate. Instead, we fit our data usinga gamma distribution, which best describes data where the standard deviation of a randomvariable increases linearly with the mean (McCabe et al., 1989), which was evident in our data.A regression model for gamma distributed data is a generalized linear model and the model isfitted by maximum likelihood. The Wald (W) statistic is used with this model to test whethera parameter is zero by comparison to the 95%-quantile of the t-distribution (Kang et al.,2004). These statistics were carried out using Statistica version 6 (StatSoft Inc, Tulsa, OK).The significance level was set at α = 0.05 (two-tailed).Chen et al.Page 4Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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RESULTSBlood ethanol concentration (BEC) values were carefully matched among the ethanol treatedD2 and B6 strain mice used in these studies (mean BEC ± SEM values were 1.64 ± 0.09 and1.64 ± 0.12 mg of ethanol/ml of blood, respectively). The mice were not tested for ethanolwithdrawal to avoid the potential confound of evoked convulsions on c-Fos immunostaining.Table 1 summarizes the number of c-Fos positive neurons in ethanol-withdrawn and control(saline) mice from the D2 and B6 inbred mouse strains across 34 brain regions emphasizingcortical, limbic, and basal ganglia circuitry. In all cases showing a strain × treatment interaction,except for lateral globus pallidus, post-hoc analysis confirmed that c-Fos induction was greaterin ethanol withdrawn D2 vs. B6 mice (Table 1). All of these regions are part of the extendedlimbic system. Notably, neuronal activation associated with ethanol withdrawal was moreintense in the hippocampal formation (dentate gyrus and CA3), amygdala and septum. Figure1 illustrates the extended limbic circuit, highlighting the connectivity of the extended limbicstructures, based upon anatomical and electrophysiological studies. Representativeimmunohistochemical results for selected brain regions are shown as follows: the hippocampalformation (Fig 2), central and basolateral amygdala (Fig 3), and septum (Fig 4).Hippocampal formationSignificant main effects of strain, treatment (ethanol withdrawal) and/or strain × treatmentinteractions were identified in three of the four regions of the hippocampal formation assessed(Table 1). Among the 34 brain regions evaluated, the most dramatic genotype-dependent neuralactivation in ethanol withdrawn mice was observed in the dentate gyrus where significanteffects of strain (W3,27=9.3, p<0.005), treatment (W3,27=13.0, p<0.0005), and strain × treatmentinteraction (W3,27=12.9, p<0.0005) were apparent. A significant effect of treatment (W3,27=9.4,p<0.005) and strain × treatment interaction (W3,27=5.3, p<0.05) were apparent in the CA3 field,and a significant effect of strain in the CA2 field (W3,27=4.2, p<0.05). There were no significanteffects detected in CA1.Extended amygdalaA main effect of treatment was apparent in six of eight regions of the extended amygdalaevaluated in the present analyses (Table 1). Significant effects of strain (W3,27=8.9, p<0.005),treatment (W3,27=9.4, p<0.005), and a strain × treatment interaction (W3,27=8.7 p<0.005) wereapparent in the basolateral amygdala. The medial central nucleus of the amygdala showed asignificant effect of treatment (W3,27=5.1, p<0.05) and a significant strain × treatmentinteraction (W3,27=7.2, p<0.005). In the lateral and capsular divisions of the central nucleus ofthe amygdala there were significant main effects of treatment (W3,27=8.3, p<0.005;W3,27=6.85,p<0.01), respectively. The basomedial amygdala showed a significant effect of treatment(W3,27=7.6, p<0.01). In addition, there were also significant effects of treatment in the bednucleus of the stria terminalis and the nucleus accumbens core (W3,27=5.0 and W3,27=4.2,p<0.05), but not the nucleus accumbens shell.SeptumA significant effect of treatment (W3,27=4.1, p<0.05) and strain × treatment interaction(W3,27=6.0, p<0.05) were apparent in the lateral septum. Post-hoc analysis confirmed asignificant induction of c-Fos in ethanol withdrawn D2 but not B6 mice (Table 1). No strainor treatment effects were detected in the medial septum.Cerebral cortexA strain × treatment effect was found in only one of the nine cortical regions evaluated, theprelimbic cortex (W3,27=4.2, p<0.05; Table 1). Significant treatment effects were apparent inChen et al.Page 5Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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the entorhinal, posterier parietal and auditory cortices (W3,27=8.1, p<0.001; W3,27=10.5,p<0.005, W3,27=4.6, p<0.05), respectively (Table 1).Basal gangliaSignificant strain × treatment interactions were identified in the substantia nigra (W3,27=5.3,p<0.05) and the lateral globus pallidus (W3,27=8.1, p<0.01). Significant treatment effects wereapparent in the caudate putamen (W3,27=13.5, p<0.0005), ventral pallidum (W3,27=6.3,p<0.01), medial globus pallidus (W3,27=4.4, p<0.05) and STN (W3,27=6.4, p<0.05). Post-hocanalysis showed significantly more c-Fos positive neurons in the lateral globus pallidus ofethanol withdrawal B6 mice. Of the 34 brain regions we examined, this is the only region thatshowed significantly greater c-Fos activation in ethanol withdrawn B6 vs. D2 mice.Hypothalamus and thalamusThe paraventricular nucleus of the hypothalamus showed significant main effects of strain(W3,27=10.8, p<0.001) and treatment (W3,27=6.5, p=0.01). No significant effects were detectedin the anterior paraventricular nucleus of the thalamus or the mediodorsal thalamus (Table 1).Midbrain and hindbrainIn the ventral tegmental area there were significant effects of strain (W3,27=4.6, p<0.05) andtreatment (W3,27=16.1, p<0.0001) but no strain × treatment interaction. The Edinger-Wesphalnucleus showed a significant main effect of treatment (W3,27=15.2, p<0.0001), but no maineffect of strain or strain × treatment interaction.DISCUSSIONCurrently, the structures responsible for the onset, propagation, and cessation of generalizedalcohol withdrawal convulsions are not known. Work in our laboratory has focused onidentifying genes that contribute to genetically determined differences in predisposition tophysical dependence and associated withdrawal following chronic ethanol exposure (Buck etal., 2002) and the neural circuitry by which the protein products of these genes influence ethanoldependence and withdrawal. The present studies address the latter using robust animal models,and are the first to begin to elucidate neural circuitry involved in genetically determineddifferences in chronic ethanol withdrawal severity. Our results suggest that behavioraldifferences between well-established mouse models of severe (D2) and mild (B6) chronicethanol withdrawal are influenced by an extended limbic circuit, including the dentate gyrusand CA3 field of the hippocampal formation as well as the extended amygdala (basolateral andcentral nucleus), lateral septum, and prelimbic cortex in genotype-dependent differences inchronic ethanol withdrawal severity. Our findings lay the foundation to elucidate the neuralmechanisms underlying the influence of individual QTLs on chronic ethanol withdrawal byregulating the activities in one or more of the identified brain regions and neurocircuits.The region with the greatest degree of neuronal activation in ethanol withdrawal D2 mice wasthe dentate gyrus of the hippocampal formation. Although increased c-Fos expression in thehippocampus has been reported in some studies to be associated with overt seizures followingchronic ethanol exposure (Dave et al., 1990; Wilce et al., 1994), it should be kept in mind thatnone of the mice in the present studies were tested for ethanol withdrawal signs (in order toavoid the potential confound of evoked convulsions on c-Fos immunostaining), nor werespontaneous seizures apparent in any of the animals. The dentate gyrus is composed primarilyof granule cells which project along the mossy fiber pathway and form excitatory synapses inarea CA3 of the hippocampus (Nadler, 2003), another region activated in the ethanol-withdrawn D2 mice. A reduction in seizure thresholds in models of limbic seizures is associatedwith loss of GABAergic interneurons in the hilus of the dentate gyrus, and formation of newChen et al. Page 6Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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recurrent excitatory circuits after mossy fiber sprouting (Dudek and Sutula, 2007). Similarly,a reduction in seizure threshold occurs during alcohol withdrawal and is associated withneuronal loss in the hilus of the dentate gyrus and area CA3 in rats (Scorza et al., 2003). Withinextended limbic circuitry, limbic seizures originating in the entorhinal cortex propagate to thehippocampus via the perforant path-dentate gyrus route (Avoli et al., 2002), and this circuitrymay play a similar role in ethanol withdrawal convulsions. We observed a trend for genotype-dependent activation of the entorhinal cortex in ethanol-withdrawn mice, and the prelimbiccortex, which influences dentate gyrus function through projections to the entorhinal cortex(Jones and Witter, 2007), showed significantly greater neuronal activation in D2 compared toB6 strain mice. Nuclei within the extended amygdala were also selectively activated duringalcohol withdrawal in D2 mice, with enhanced c-Fos immunoreactivity in the basolateral andmedial central nuclei of the amygdala. The amygdala is part of the medial temporal structuresof the brain associated with human partial seizures (Rogawski et al., 2003). The extendedamygdala has a role in ethanol withdrawal associated kindling, anxiety and negative affectivestates (Borlikova et al., 2006; Feng et al., 2007; Funk et al., 2006). Repeated ethanol withdrawalresults in a progressive enhancement of convulsion activity known as kindling (Ballenger andPost, 1978; Becker and Hale, 1993) and is associated with greater c-Fos activation in the thecentral nucleus and basolateral amygdala (Borlikova et al., 2006). The amygdala alsoparticipates in generating tonic convulsions during generalized audiogenic seizures in ethanolwithdrawn animals, which are attenuated by microinjection of 2-amino-7-phosphonoheptanoate (AP7), an NMDA receptor antagonist, into the central or lateral nucleiof the amygdala (Feng et al., 2007). The central nucleus of the amygdala also participates inethanol consumption and preference (Dhaher et al., 2008), and administration of a CRF1receptor antagonist administered into the central nucleus during ethanol withdrawal reducesvoluntary consumption ofethanol self-administration in alcohol-dependent animals (Funk etal., 2006).The initiation and propagation of limbic motor convulsions have been shown to involve thehippocampus and parahippocampal structures (e.g., amygdala and lateral septum; Lothman,1994; Lothman et al., 1991). Connections between these regions primarily use glutamate as aneurotransmitter, whereas GABAergic interneurons modulate this circuitry (Mraovitch andCalando, 1999). The prelimbic cortex, which also showed genotype-dependent activation inethanol withdrawn mice, sends glutamatergic projections to the hippocampus and basolateralamygdala (Mraovitch and Calando, 1999; Watson et al., 1985). Furthermore, the basolateralamygdala and the hippocampus both have excitatory glutamatergic projections (Nieuwenhuys,1996), while the lateral septum sends inhibitory GABAergic projections (Nieuwenhuys,1996), to the central nucleus of the amygdala. Alcohol exposure and withdrawal could induceneuroplasticity within this circuitry decreasing inhibitory GABAergic tone and enhancingexcitatory glutamatergic drive and leading to an exacerbation of withdrawal severity in D2compared to B6 mice. Future studies using site-directed pharmacological manipulations willbe needed to further define this circuit in ethanol withdrawal.The extended limbic circuit also may contribute to other behaviors associated with thewithdrawal syndrome (i.e., tremors, depression-like behavior, anxiety-like behavior, andemotionality). Alterations in hippocamal function are associated with depression. Bingealcohol exposure decreases neurogenesis in the dentate gyrus (He et al., 2005; Herrera et al.,2003; Nixon and Crews, 2002), which is thought to be involved in the therapeutic efficacy ofantidepressant treatment (Kempermann et al., 2008). Notably, D2 mice exhibit more severeethanol withdrawal-associated depression-like behavior than B6 animals (L. Milner and K.Buck, unpublished results). Anxiety is another sign of ethanol withdrawal, and prelimbiccortical lesions increase anxiety-like behavior in rats (Jinks and McGregor, 1997). While manysigns of the ethanol withdrawal syndrome are genetically correlated with convulsion (e.g., HIC)severity (i.e., tremors, hypoactivity, emotionality; Belknap et al., 1987; Feller et al., 1994;Chen et al.Page 7Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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Kosobud and Crabbe, 1986) others are not (i.e., tail stiffness; Kosobud and Crabbe, 1986). Itis well established that the amygdala plays a critical role in anxiety-like behavior in ethanolwithdrawn animals (Koob and Le Moal, 2008). Additionally, it is well-documented that alcoholwithdrawal HIC severity is genetically correlated with alcohol preference drinking (Hitzemannet al., 2008; Metten and Crabbe, 2005; Metten et al., 1998). The central nucleus of the amygdalahas a prominent role in alcohol preference (Dhaher et al., 2008) and projects to the extendedlimbic nuclei i.e., dentate gyrus. Future studies will assess the contribution of this circuit to therelationship between ethanol preference and withdrawal.Previously, we identified an extended basal ganglia circuit which is activated in acute ethanolwithdrawn D2 mice (Kozell et al., 2005). This acute withdrawal paradigm represents the initialsensitivity to neural excitability following withdrawal from a single hypnotic dose of ethanol(4 g/kg, ip). In the present study the induction of ethanol dependence following chronicexposure to ethanol vapor shows activation of different brain circuitry and involves limbicstructures in which activation was not apparent using the acute model. For example, in thepresent study using the chronic model, prominent activation was evident in ethanol-withdrawnD2 mice in the hippocampal formation, amygdala, septum and prelimbic cortex. In the acutemodel, there was only slight activation in these regions in ethanol-withdrawn D2 mice,contrasting with prominent activation within the basal ganglia, including the substantia nigrapars reticulata, subthalamic nucleus and ventral pallidum (Kozell et al., 2005). More recently,our laboratory has shown that lesions of the lateral substantia nigra pars reticulata, but not thesubthalamic nucleus, reduce withdrawal severity following both acute and repeated ethanolexposures (Chen et al., 2008). The difference in the pattern of neural activation in acute vs.chronic ethanol withdrawal may reflect neuroadaptations that occur with the transition fromacute physiological dependence and associated withdrawal to more severe dependence andwithdrawal after chronic ethanol exposure. Additionally, previous study has shown a shift fromc-Fos to other immediate early gene products (e.g., deltaFosB) in discrete brain regions whendrugs are administrated from acutely to chronically, and this shift is involved in epigenicmodification (Hope et al., 1994; Kumar et al., 2005; Young et al., 1991). This may also occurfor the activation of basal ganglia circuit in acute and chronic ethanol withdrawal. It remainsto be determined how enduring neuronal activation is in the acute and chronic models. It willbe important for future studies to examine additional time points after withdrawal to addressthis issue, and better understand the neuroadaptations that occur from initial drug use to laterstages of dependence (Kalivas and Volkow, 2005).In summary, the present study, which assessed the pattern of neural activation associated withchronic ethanol withdrawal, points to the involvement of limbic circuitry in geneticallydetermined differences in chronic ethanol withdrawal. Our results lay the groundwork forfurther characterization of this circuit in relation to ethanol dependence. Potential future studiesto address whether neuronal activation in limbic circuitry is essential to chronic ethanolwithdrawal severity include assessing neuronal activation using F2 intercross, heterogeneousstock (HS), and/or short-term selective breeding populations that differ in chronic ethanolwithdrawal severity. The latter would be particularly useful because sufficient numbers ofanimals could be bred to assess the time course of c-Fos induction, as well as that of otherimmediate early gene products (e.g., ΔFOSB) associated with physiological dependence onethanol and associated withdrawal. Finally, future site-directed lesion and neurochemicalstudies will be necessary to determine the influence of limbic nuclei and pathways on chronicethanol withdrawal severity.Chen et al. Page 8Alcohol. Author manuscript; available in PMC 2010 September 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript